Showing papers on "Antimicrobial peptides published in 2009"

Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations

Abstract: An increasing amount of information on the action of antimicrobial peptides (AMPs) at the molecular level has not yet been translated into a comprehensive understanding of effects in bacteria. Although some biophysical attributes of AMPs have been correlated with macroscopic features, the physiological relevance of other properties has not yet been addressed. Pertinent and surprising conclusions have therefore been left unstated. Strong membrane-binding and micromolar therapeutic concentrations of AMPs indicate that membrane-bound concentrations may be reached that are higher than intuitively expected, triggering disruptive effects on bacteria.

Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent.

Abstract: Antimicrobial cationic peptides are of interest because they can combat multi-drug-resistant microbes. Most peptides form alpha-helices or beta-sheet-like structures that can insert into and subsequently disintegrate negatively charged bacterial cell surfaces. Here, we show that a novel class of core-shell nanoparticles formed by self-assembly of an amphiphilic peptide have strong antimicrobial properties against a range of bacteria, yeasts and fungi. The nanoparticles show a high therapeutic index against Staphylococcus aureus infection in mice and are more potent than their unassembled peptide counterparts. Using Staphylococcus aureus-infected meningitis rabbits, we show that the nanoparticles can cross the blood-brain barrier and suppress bacterial growth in infected brains. Taken together, these nanoparticles are promising antimicrobial agents that can be used to treat brain infections and other infectious diseases.

The Roles of Antimicrobial Peptides in Innate Host Defense

Abstract: Antimicrobial peptides (AMPs) are multi-functional peptides whose fundamental biological role in vivo has been proposed to be the elimination of pathogenic microorganisms, including Gram-positive and -negative bacteria, fungi, and viruses. Genes encoding these peptides are expressed in a variety of cells in the host, including circulating phagocytic cells and mucosal epithelial cells, demonstrating a wide range of utility in the innate immune system. Expression of these genes is tightly regulated; they are induced by pathogens and cytokines as part of the host defense response, and they can be suppressed by bacterial virulence factors and environmental factors which can lead to increased susceptibility to infection. New research has also cast light on alternative functionalities, including immunomodulatory activities, which are related to their unique structural characteristics. These peptides represent not only an important component of innate host defense against microbial colonization and a link between innate and adaptive immunity, but also form a foundation for the development of new therapeutic agents.

Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides.

Abstract: There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other.

Use of Artificial Intelligence in the Design of Small Peptide Antibiotics Effective against a Broad Spectrum of Highly Antibiotic-Resistant Superbugs

Abstract: Increased multiple antibiotic resistance in the face of declining antibiotic discovery is one of society's most pressing health issues. Antimicrobial peptides represent a promising new class of antibiotics. Here we ask whether it is possible to make small broad spectrum peptides employing minimal assumptions, by capitalizing on accumulating chemical biology information. Using peptide array technology, two large random 9-amino-acid peptide libraries were iteratively created using the amino acid composition of the most active peptides. The resultant data was used together with Artificial Neural Networks, a powerful machine learning technique, to create quantitative in silico models of antibiotic activity. On the basis of random testing, these models proved remarkably effective in predicting the activity of 100,000 virtual peptides. The best peptides, representing the top quartile of predicted activities, were effective against a broad array of multidrug-resistant "Superbugs" with activities that were equal to or better than four highly used conventional antibiotics, more effective than the most advanced clinical candidate antimicrobial peptide, and protective against Staphylococcus aureus infections in animal models.

De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers

Abstract: The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.

Broad-spectrum antimicrobial peptides by rational combinatorial design and high-throughput screening: the importance of interfacial activity.

Abstract: We recently described ten peptides selected from a 16,384-member combinatorial library based on their ability to permeabilize synthetic lipid vesicles in vitro (Rathinakumar R and Wimley WC, J. Am. Chem. Soc. 2008, 130, 9849-9858). These peptides did not share a common sequence motif, length or net charge; nonetheless they shared a mechanism of action that is similar to the natural membrane permeabilizing antimicrobial peptides (AMP). To characterize the selected peptides and to compare the activity of AMPs in vivo and in vitro we report on the biological activity of the same selected peptides in bacteria, fungi, and mammalian cells. Each of the peptides has sterilizing activity against all classes of microbes tested, at 2-8 μM peptide, with only slight hemolytic or cytotoxicity against mammalian cells. Similar to many natural AMPs, bacteria are killed within a few minutes of peptide addition and the lethal step in vivo is membrane permeabilization. Single D-amino acid substitutions eliminated or diminished the secondary structure of the peptides and yet they retained activity against some microbes. Thus, secondary structure and biological activity are not coupled, consistent with the hypothesis that AMPs do not form pores of well defined structure in membranes, but rather destabilize membranes by partitioning into membrane interfaces and disturbing the organization of the lipids, a property that we have called “interfacial activity”. The observation that broad-spectrum activity, but not all antimicrobial activity, is lost by small changes to the peptides suggests that the in vitro screen is specifically selecting for the rare peptides that have broad-spectrum activity. We put forth the hypothesis that methods focusing on screening peptide libraries in vitro for members with the appropriate interfacial activity can enable the design, selection and discovery of novel, potent and broad-spectrum membrane-active antibiotics.

Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps

Abstract: The formation of extracellular traps (ETs) by phagocytic cells has been recognized as a novel and important mechanism of the host innate immune response against infections. ETs are formed by different host immune cells such as neutrophils, mast cells, and eosinophils after stimulation with mitogens, cytokines, or pathogens themselves, in a process dependent upon induction of a reactive-oxygen-species-mediated signaling cascade. ETs consist of nuclear or mitochondrial DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases and thereby provide a matrix to entrap and kill microbes and to induce the contact system. This review summarizes the latest research on ETs and their role in innate immunity and host innate defense. Attention is also given to mechanisms by which certain leading bacterial pathogens have evolved to avoid entrapment and killing in these specialized structures.

PhytAMP: a database dedicated to antimicrobial plant peptides

Abstract: Plants produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens Based on amino acid sequence homology, these peptides were classified mostly as α-defensins, thionins, lipid transfer proteins, cyclotides, snakins and hevein-like Although many antimicrobial plant peptides are now well characterized, much information is still missing or is unavailable to potential users The compilation of such information in one centralized resource, such as a database would therefore facilitate the study of the potential these peptide structures represent, for example, as alternatives in response to increasing antibiotic resistance or for increasing plant resistance to pathogens by genetic engineering To achieve this goal, we developed a new database, PhytAMP, which contains valuable information on antimicrobial plant peptides, including taxonomic, microbiological and physicochemical data Information is very easy to extract from this database and allows rapid prediction of structure/function relationships and target organisms and hence better exploitation of plant peptide biological activities in both the pharmaceutical and agricultural sectors PhytAMP may be accessed free of charge at http://phytamppfba-laborg

Anionic Antimicrobial Peptides from Eukaryotic Organisms

Abstract: Anionic antimicrobial peptides / proteins (AAMPs) were first reported in the early 1980s and since then, have been established as an important part of the innate immune systems of vertebrates, invertebrates and plants. These peptides are active against bacteria, fungi, viruses and pests such as insects. AAMPs may be induced or expressed constitutively and in some cases, antimicrobial activity appears to be a secondary role for these peptides with other biological activities constituting their primary role. Structural characterization shows AAMPs to generally range in net charge from -1 to -7 and in length from 5 residues to circa 70 residues and for a number of these peptides, post-translational modifications are essential for antimicrobial activity. Membrane interaction appears key to the antimicrobial function of AAMPs and to facilitate these interactions, these peptides generally adopt amphiphilic structures. These architectures vary from the alpha-helical peptides of some amphibians to the cyclic cystine knot structures observed in some plant proteins. Some AAMPs appear to use metal ions to form cationic salt bridges with negatively charged components of microbial membranes, thereby facilitating interaction with their target organisms, but in many cases, the mechanisms underlying the antimicrobial action of these peptides are unclear or have not been elucidated. Here, we present an overview on current research into AAMPs, which suggests that these peptides are an untapped source of putative antimicrobial agents with novel mechanisms of action and possess potential for application in the medical and biotechnological arenas.

Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides with No Influence upon the Activity Spectrum

Abstract: Early studies of immobilized peptides mainly focused upon the relationship between structural properties and the activity of soluble and surface-tethered sequences. The intention of this study was to analyze the influence of immobilization parameters upon the activity profile of peptides. Resin beads (TentaGel S NH(2), HypoGel 400 NH(2), and HypoGel 200 NH(2)) with polyethylene glycol spacers of different lengths were rendered antimicrobial by linkage of an amphipathic model KLAL peptide and magainin-derived MK5E. Standard solid-phase peptide synthesis, thioalkylation, and ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions. Depending upon the resin capacity and the coupling strategies, peptide loading ranged between 0.1 and 0.25 micromol/mg for C-terminally and around 0.03 micromol/mg for N-terminally and side-chain-immobilized peptides. Tethering conserved the activity spectra of the soluble peptides at reduced concentrations. The resin-bound peptides were antimicrobial toward Escherichia coli and Bacillus subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. B. subtilis was more susceptible than E. coli. The antimicrobial activity distinctly decreased with reduction of the spacer length. Slight differences in the antimicrobial effect of KLAL and MK5E bound at different chain positions on TentaGel S NH(2) suggest that the activity is less dependent upon the position of immobilization. Soluble KLAL was active toward red blood cells, whereas MK5E was nonhemolytic at up to about 400 microM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH(2)-bound peptides enhanced the permeability of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline) and mixed POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG) bilayers used to model the charge properties of the biological targets. The results suggest that surface immobilization of the cationic amphipathic antimicrobial peptides does not influence the membrane-permeabilizing mode of action. Peptide insertion into the target membrane and likely the exchange of membrane-stabilizing bivalent cations contribute to the antimicrobial effect. In conclusion, reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters, with the length of the spacer and the amount of target-accessible peptide being the most important factors.

Anopheles gambiae PGRPLC-Mediated Defense against Bacteria Modulates Infections with Malaria Parasites

Abstract: Recognition of peptidoglycan (PGN) is paramount for insect antibacterial defenses. In the fruit fly Drosophila melanogaster, the transmembrane PGN Recognition Protein LC (PGRP-LC) is a receptor of the Imd signaling pathway that is activated after infection with bacteria, mainly Gram-negative (Gram−). Here we demonstrate that bacterial infections of the malaria mosquito Anopheles gambiae are sensed by the orthologous PGRPLC protein which then activates a signaling pathway that involves the Rel/NF-κB transcription factor REL2. PGRPLC signaling leads to transcriptional induction of antimicrobial peptides at early stages of hemolymph infections with the Gram-positive (Gram+) bacterium Staphylococcus aureus, but a different signaling pathway might be used in infections with the Gram− bacterium Escherichia coli. The size of mosquito symbiotic bacteria populations and their dramatic proliferation after a bloodmeal, as well as intestinal bacterial infections, are also controlled by PGRPLC signaling. We show that this defense response modulates mosquito infection intensities with malaria parasites, both the rodent model parasite, Plasmodium berghei, and field isolates of the human parasite, Plasmodium falciparum. We propose that the tripartite interaction between mosquito microbial communities, PGRPLC-mediated antibacterial defense and infections with Plasmodium can be exploited in future interventions aiming to control malaria transmission. Molecular analysis and structural modeling provided mechanistic insights for the function of PGRPLC. Alternative splicing of PGRPLC transcripts produces three main isoforms, of which PGRPLC3 appears to have a key role in the resistance to bacteria and modulation of Plasmodium infections. Structural modeling indicates that PGRPLC3 is capable of binding monomeric PGN muropeptides but unable to initiate dimerization with other isoforms. A dual role of this isoform is hypothesized: it sequesters monomeric PGN dampening weak signals and locks other PGRPLC isoforms in binary immunostimulatory complexes further enhancing strong signals.

Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence.

Abstract: Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydra's epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence. (C) 2008 Elsevier Ltd. All rights reserved.

Mucosal Gene Expression of Antimicrobial Peptides in Inflammatory Bowel Disease Before and After First Infliximab Treatment

Abstract: Background Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD.

Protease inhibitors from plants with antimicrobial activity.

Abstract: Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

Swarming motility: a multicellular behaviour conferring antimicrobial resistance

Abstract: Swarming is a type of social motility allowing the migration of highly differentiated bacterial cells. Swarming shares many similarities with biofilm communities, which are notable for their high resistance to antimicrobial agents. We investigate here if the swarming behaviour could also be associated with a widespread antimicrobial resistant phenotype. Challenged with 13 antibiotics from various classes, swarm cells of Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Burkholderia thailandensis and Bacillus subtilis showed higher resistance than their planktonic counterparts to all the antibiotics tested, except for the antimicrobial peptides. Using P. aeruginosa as a model, this multiresistant phenotype was shown to be transient and intrinsically linked to the swarming state. Resistance of swarm cells towards other antimicrobial agents, such as triclosan and a heavy metal (arsenite), was also observed. Neither RND-type efflux pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, nor a biofilm-associated resistance mechanism involving periplasmic glucans, appear to account for the resistance of swarm cells. Together with the high resistance of biofilms, these results support the hypothesis that antimicrobial resistance is a general feature of bacterial multicellularity. Swarming motility might thus represent a form of social behaviour useful as a model to investigate biofilm antibiotic resistance.

Synergistic Interaction between Silver Nanoparticles and Membrane-Permeabilizing Antimicrobial Peptides

Abstract: Silver nanoparticles, as well as antimicrobial peptides (AMPs), can be used to fight infectious diseases. Since AMPs are known to permeabilize bacterial membranes and might therefore help silver nanoparticles to access internal target sites, we investigated their combined activities and showed synergistic effects between polymyxin B and silver nanoparticles for gram-negative bacteria.

Multifunctional host defense peptides: antimicrobial peptides, the small yet big players in innate and adaptive immunity.

Abstract: The term 'antimicrobial peptides' refers to a large number of peptides first characterized on the basis of their antibiotic and antifungal activities. In addition to their role as endogenous antibiotics, antimicrobial peptides, also called host defense peptides, participate in multiple aspects of immunity (inflammation, wound repair, and regulation of the adaptive immune system) as well as in maintaining homeostasis. The possibility of utilizing these multifunctional molecules to effectively combat the ever-growing group of antibiotic-resistant pathogens has intensified research aimed at improving their antibiotic activity and therapeutic potential, without the burden of an exacerbated inflammatory response, but conserving their immunomodulatory potential. In this minireview, we focus on the contribution of small cationic antimicrobial peptides - particularly human cathelicidins and defensins - to the immune response and disease, highlighting recent advances in our understanding of the roles of these multifunctional molecules.

Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37

Abstract: Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.

DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses

Abstract: Generation of microbicidal reactive oxygen species (ROS) is a pivotal protective component of the innate immune system in many eukaryotes. NOD (nucleotide oligomerisation domain containing protein)-like receptors (NLRs) have been implicated as phylogenetically ancient sensors of intracellular pathogens or endogenous danger signals. NOD2 recognizes the bacterial cell wall component muramyldipeptide leading to NF kappa B and MAPK activation via induced proximity signalling through the serine-threonine kinase RIP2. In addition to the subsequent induction of cytokines and antimicrobial peptides, NOD2 has been shown also to exert a direct antibacterial effect. Using a fluorescence-based ROS detection assay we demonstrate controlled ROS generation as an integral component of NOD2-induced signalling in epithelial cells. We demonstrate that the NAD(P)H oxidase family member DUOX2 is involved in NOD2-dependent ROS production. Coimmunoprecipitation and fluorescence microscopy were used to show that DUOX2 interacts and colocalizes with NOD2 at the plasma membrane. Moreover, simultaneous overexpression of NOD2 and DUOX2 was found to result in cooperative protection against bacterial cytoinvasion using the Listeria monocytogenes infection model. RNAi-based studies revealed that DUOX2 is required for the direct bactericidal properties of NOD2. Our results demonstrate a new role of ROS as effector molecules of protective cellular signalling in response to a defined danger signal carried out by a mammalian intracellular NLR system.

Activation of the human contact system on neutrophil extracellular traps.

Abstract: Pattern recognition is an integral part of the innate immune system. The human contact system has been shown to interact with the surface of many bacterial and fungal pathogens, and once activated leads to the generation of antimicrobial peptides and the proinflammatory mediator bradykinin. Here we show that apart from these surfaces also neutrophil extracellular traps (NETs) provide a surface that allows the binding and activation of the contact system. In addition, we present evidence that M1 protein, a streptococcal surface protein, in concert with human fibrinogen triggers polymorphonuclear neutrophils to form NETs.

Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis.

Abstract: After being infected by the fungus Drechmeria coniospora, Caenorhabditis elegans produces antimicrobial peptides in its epidermis, some regulated by a signaling cascade involving a p38 mitogen-activated protein kinase. Here we show that infection-induced expression of peptides of the Caenacin family occurred independently of the p38 pathway. The caenacin (cnc) genes enhanced survival after fungal infection, and neuronal expression of the transforming growth factor-beta homolog DBL-1 promoted cnc-2 expression in the epidermis in a dose-dependent paracrine way. Our results lead to a model in which antifungal defenses are coordinately regulated by a cell-autonomous p38 cascade and a distinct cytokine-like transforming growth factor-beta signal from the nervous system, each of which controls distinct sets of antimicrobial peptide-encoding genes in the epidermis.